Streptococcus pneumoniae is a widespread pathogen and a leading cause of pneumonia, otitis media, bacteremia and meningitis. Since the current multivalent polysaccharide vaccines fail to protect those most susceptible to infection (children under the age of 2 and older adults), identification of proteins important to pneumococcal virulence is currently a high priority for new vaccine initiatives. The emergence and global spread of penicillin resistant pneumococci has also focused interest on horizontal gene transfer by natural transformation. We have recently reported the first use of random translational gene fusions (PhoA mutagenesis) to identify and alter exported proteins in a gram positive organism. We have also developed random transcriptional fusions (LacZ mutagenesis) to assess gene regulation in pneumococcus. Using this new technology, we have characterized several genetic loci with sequence similarity to known families of exported proteins. Homologs were found to protein-dependent peptide permeases, penicillin binding proteins, Clp proteases, two- component sensor regulators and ABC (ATP binding cassette) transporters responsible for the export of the RTX class of bacterial toxins. This technology makes it possible for the first time to map the surface proteins of the pneumococcus in a systematic and complete manner. Given the wealth of new proteins identified by our new gene fusion technology, we choose to focus this project on the identification and characterization of those surface proteins which are: 1] virulence determinants and thus could serve as candidates for a conjugate vaccine and 2] participants in the process of transformation by exogenous DNA. To do so, we will assess our banks of mutants for a loss of function in the steps of pneumococcal infection, particularly adherence to epithelia and endothelia and in the process of transformation. Based on preliminary work, mutations in 5 loci decrease pneumococcal adhesion by about 60% to Type II lung cells or endothelial cells. We will determine by genetic and molecular analysis if these loci directly encode adhesions or if they mediate expression as part of a regulatory cascade. The impact of these loci on virulence will be assessed in animal models for colonization and infection. Surface proteins that participate in adherence or other steps in pathogenesis will be assessed for immunogenic and protective activity with the aim of identifying new protein vaccine candidates. In further preliminary work, two distinct mutations decrease the efficiency of natural transformation by about 90. These loci are plpA which encodes a peptide permease and rec which is an operon that encodes an exported protein and a RecA homolog. We will test the hypothesis that plpA is a regulatory locus modulating transformation by transporting small oligopeptides that serve as intracellular messengers. The rec operon, is upregulated during transformation and is the first transformation-regulated gene to be cloned. We will identify both cis and trans acting elements that regulate expression of this locus in order to begin to assemble a model of the signaling cascade that controls the process of transformation.